Harmful algae and their potential impacts on the coastal ecosystem: growth and toxin production dynamics

The main goal of the present thesis was to study some harmful algal species which cause blooms in Italian coastal waters, leading to consequences for human health, coastal ecosystem, fishery and tourism. In particular, in the first part of this thesis the toxicity of Adriatic strains of the raphidophyte Fibrocapsa japonica was investigated. Despite several hypotheses have been proposed for the toxic mechanism of the raphidophytes, especially for the species Chattonella antiqua and C. marina, which have been studied more extensively, just a few studies on the toxic effects of these species for different organisms were reported. Moreover, a careful reading of the literature evidenced as any ichthyotoxic events reported worldwide can be linked to F. japonica blooms. Although recently several studies were performed on F. japonica strains from the USA, Japan, Australia, New Zealand, the Netherlands, Germany, and France in order to characterize their growth and toxicity features, the work reported in this thesis results one of the first investigation on the toxic effects of F. japonica for different organisms, such as bacteria, crustaceans and fish. Mortality effects, together with haemolysis of fish erythrocytes, probably due to the relatively high amount of PUFAs produced by this species, were observed. Mortality for fish, however, was reported only at a high cell density and after a long exposition period (9-10 days); moreover a significant increase of H2O2 obtained in the tanks where sea basses were exposed to F. japonica was also relevant. This result may justify the absence of ichthyotoxic events in the Italian coasts, despite F. japonica blooms detected in these areas were characterized by high cell densities. This work reports also a first complete characterization of the fatty acids produced and extracellularly released by the Adriatic F. japonica, and results were also compared with the fatty acid profile of other strains. The absence of known brevetoxins in F. japonica algal extracts was also highlighted, leading to the hypothesis that the toxicity of F. japonica may be due to a synergic effect of PUFAs and ROS. Another microalgae that was studied in this thesis is the benthic dinoflagellate Ostreopsis cf. ovata. This species was investigated with the aim to investigate the effect of environmental parameters on its growth and toxicity. O. cf. ovata, in fact, shows different blooming periods along the Italian coasts and even the reported toxic effects are variable. The results of this work confirmed the high variability in the growth dynamic and toxin content of several Italian strains which were isolated in recent years along the Adriatic and Tyrrhenian Seas. Moreover, the effects of temperature and salinity on the behaviour of the different isolates are in good agreement with the results obtained from field surveys, which evidence as the environmental parameters are important factors modulating O. cf. ovata proliferation. Another relevant result that was highlighted is the anomaly in the production of palytoxin-like compounds reported by one of the studied isolate, in particular the one isolated in 2008 in Ancona (Adriatic Sea). Only this strain reported the absence of two (ovatoxin-b and –c) of the five ovatoxins so far known in the toxin profile and a different relative abundance of the other toxins. The last aspect that was studied in this thesis regards the toxin biosythesis. In fact, toxins produced (palytoxin-like compounds) or supposed to be produced (brevetoxin-like compounds) by O. cf. ovata and F. japonica, respectively, are polyketides, which are highly oxygenated compounds synthesized by complex enzymes known as polyketide synthase (PKS) enzymes. These enzymes are multi-domain complexes that structurally and functionally resemble the fatty acid synthases (FASs). This work reports the first study of PKS proteins in the dinoflagellates O. cf. ovata, C. monotis and in the raphidophyte F. japonica. For the first time some PKSs were identified in these species, confirming the presence of PKS proteins predicted by the in silico translation of the transcripts found in K. brevis also in other species. The identification of O. cf. ovata PKSs and the localization of the palytoxin-like compounds produced by this dinoflagellate in a similar location (chloroplast) as that observed for other dinoflagellate and cyanobacterial toxins provides some indication that these proteins may be involved in polyketide biosynthesis. However, their potential function as fatty acid synthases cannot be ruled out, as plant fatty acid synthesis also occurs within chloroplasts. This last hypothesis is also supported by the fact that in all the investigated species, and in particular in F. japonica, PKS proteins were present. Therefore, these results provide an important contribution to the study of the polyketides and of the involvement of PKS proteins in the toxin biosynthesis

Spring composition of the macroalgal vegetation of a small offshore island in the north-western Mediterranean (Gallinara Island, Ligurian Sea)

Gallinara Island, a small island located 1.5 km off the shore of Liguria (Italy, north-western Mediterranean Sea) was included in a list of proposed Marine Protected Areas (MPA) in the early 90s. Since then, its benthic assemblages have been studied in detail and the main macrophytic communities have been mapped. A detailed assessment of its benthic macroalgal flora, however, has never been made. Gallinara was visited in the course of 5 consecutive years and its macroalgal flora was studied based on collections made by snorkelling and SCUBA diving. Overall, 141 macroalgal taxa were collected and identified (23 Chlorophyta, 94 Rhodophyta, 24 Ochrophyta); 91 of them represent new records for the island. One of the most notable new records is the non-indigenous red alga Womersleyella setacea, previously unreported from the island and widely distributed, particularly on the south-eastern shore. Observations made in the course of the surveys confirm the rarefaction of some large-sized brown algae (particularly Sargassum vulgare) but indicate also that others previously reported as rare (Cystoseira compressa, Dictyopteris polypodioides) are still common on the island

Toxin Levels and Profiles in Microalgae from the North-Western Adriatic Sea—15 Years of Studies on Cultured Species

The Northern Adriatic Sea is the area of the Mediterranean Sea where eutrophication and episodes related to harmful algae have occurred most frequently since the 1970s. In this area, which is highly exploited for mollusk farming, the first occurrence of human intoxication due to shellfish consumption occurred in 1989, nearly 10 years later than other countries in Europe and worldwide that had faced similar problems. Until 1997, Adriatic mollusks had been found to be contaminated mostly by diarrhetic shellfish poisoning toxins (i.e., okadaic acid and dinophysistoxins) that, along with paralytic shellfish poisoning toxins (i.e., saxitoxins), constitute the most common marine biotoxins. Only once, in 1994, a toxic outbreak was related to the occurrence of paralytic shellfish poisoning toxins in the Adriatic coastal waters. Moreover, in the past 15 years, the Adriatic Sea has been characterized by the presence of toxic or potentially toxic algae, not highly widespread outside Europe, such as species producing yessotoxins (i.e., Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum), recurrent blooms of the potentially ichthyotoxic species Fibrocapsa japonica and, recently, by blooms of palytoxin-like producing species of the Ostreopsis genus. This review is aimed at integrating monitoring data on toxin spectra and levels in mussels farmed along the coast of the Emilia-Romagna region with laboratory studies performed on the species involved in the production of those toxins; toxicity studies on toxic or potentially toxic species that have recently appeared in this area are also reviewed. Overall, reviewed data are related to: (i) the yessotoxins producing species P. reticulatum, G. spinifera and L. polyedrum, highlighting genetic and toxic characteristics; (ii) Adriatic strains of Alexandrium minutum, Alexandrium ostenfeldii and Prorocentrum lima whose toxic profiles are compared with those of strains of different geographic origins; (iii) F. japonica and Ostreopsis cf. ovata toxicity. Moreover, new data concerning domoic acid production by a Pseudo-nitzschia multistriata strain, toxicity investigations on a Prorocentrum cf. levis, and on presumably ichthyotoxic species, Heterosigma akashiwo and Chattonella cf. subsalsa, are also reported

Dinoflagellate resting cysts from surface sediments of the Adriatic Ports: distribution and potential spreading patterns

The ability of microalgae to preserve viable in coastal sediments as resting forms provides a reservoir of biodiversity and a useful tool to determine species spreadings. This study represents the first port baseline survey on dinoflagellate cysts, investigated in nine Adriatic ports during a cross border project. 40 dinoflagellate taxa were detected. The assemblages resulted in all ports dominated by Lingulodinium polyedra and Alexandrium minutum/affine/tamutum group. General separation to the western and eastern side of the Adriatic regarding cysts assemblage composition, partially abundance, was observed. Seven taxa were detected as non-indigenous species for the Adriatic. Two taxa are included in the list of harmful aquatic organisms, indicating the potential threat of ballast waters in the Adriatic. Potential spreading of taxa by general circulation and ballast waters, intra- and extra-Adriatic was investigated. The entering in to force of the ballast waters management regulations should enhance prospects to minimize future harmful impacts

Microbial dynamics during harmful dinoflagellate Ostreopsis cf. ovata growth: Bacterial succession and viral abundance pattern

Algal\u2013bacterial interactions play a major role in shaping diversity of algal associated bacterial communities. Temporal variation in bacterial phylogenetic composition reflects changes of these complex interactions which occur during the algal growth cycle as well as throughout the lifetime of algal blooms. Viruses are also known to cause shifts in bacterial community diversity which could affect algal bloom phases. This study investigated on changes of bacterial and viral abundances, bacterial physiological status, and on bacterial successional pattern associated with the harmful benthic dinoflagellate Ostreopsis cf. ovata in batch cultures over the algal growth cycle. Bacterial community phylogenetic structure was assessed by 16S rRNA gene ION torrent sequencing. A comparison between bacterial community retrieved in cultures and that one co-occurring in situ during the development of the O. cf. ovata bloom from where the algal strain was isolated was also reported. Bacterial community growth was characterized by a biphasic pattern with the highest contributions (~60%) of highly active bacteria found at the two bacterial exponential growth steps. An alphaproteobacterial consortium composed by the Rhodobacteraceae Dinoroseobacter (22.2%\u201335.4%) and Roseovarius (5.7%\u201318.3%), together with Oceanicaulis (14.2- 40.3%), was strongly associated with O. cf. ovata over the algal growth. The Rhodobacteraceae members encompassed phylotypes with an assessed mutualisticpathogenic bimodal behavior. Fabibacter (0.7%\u201325.2%), Labrenzia (5.6%\u201324.3%), and Dietzia (0.04%\u20131.7%) were relevant at the stationary phase. Overall, the successional pattern and the metabolic and functional traits of the bacterial community retrieved in culture mirror those ones underpinning O. cf. ovata bloom dynamics in field. Viral abundances increased synoptically with bacterial abundances during the first bacterial exponential growth step while being stationary during the second step. Microbial trends also suggest that viruses induced some shifts in bacterial community composition

Produzione di ovatossine nella dinoflagellata Ostreopsis cf. ovata: studi al variare di fattori abiotici e biotici

Fioriture di Ostreopsis spp. sono state osservate nell'ultimo ventennio nel Mediterraneo e la loro diffusione aumenta nel mondo ogni anno, rappresentando una potenziale minaccia per la salute umana e per l'ecosistema marino (Rhodes et al., 2011). In particolare la specie O. cf. ovata negli ultimi anni è presente in varie aree costiere europee, tra cui l'Italia. Nonostante in queste zone non siano stati registrati casi di intossicazione umana per consumo di prodotti ittici, la problematica è di grande interesse a seguito dell'elevata tossicità della palitossina e dei congeneri, denominati ovatossine, prodotti da questa dinoflagellata. Le fioriture hanno spesso mostrato differenti caratteristiche in termini di periodo di sviluppo e relative condizioni abiotiche, di densità algale e di tossicità (Cohu et al., 2013). Allo scopo di individuare i fattori abiotici (e.g. temperatura, salinità, luce) e biotici (e.g. presenza di specie algali competitrici o batteri) che maggiormente influenzano le fioriture ed il loro livello di tossicità, sono stati effettuati studi in coltura utilizzando ceppi di O. cf. ovata isolati da coste italiane, saggiando differenti condizioni. In primo luogo è stato evidenziato che le diverse ovatossine analizzate negli estratti di O. cf. ovata aumentano quantitativamente lungo la crescita (Pezzolesi et al., 2014) con un rilascio nel mezzo extracellulare maggiore in fase stazionaria. Inoltre il profilo tossinologico ha evidenziato un'alta variabilità sia in termini qualitativi (i.e. assenza di alcune ovatossine) che quantitativi (i.e. contenuto di tossina per cellula o litro di coltura) in funzione dei differenti ceppi esaminati. In generale è emerso che temperatura, salinità e concentrazione dei nutrienti sono fattori abiotici che influiscono nella produzione cellulare di tossine (Pistocchi et al., 2011; Pezzolesi et al., 2012; Vanucci et al., 2012b). Nello specifico la disponibilità di nutrienti è importante per la biosintesi di questi composti che contengono azoto e hanno alto peso molecolare. I risultati hanno evidenziato che in carenza di azoto (N), in particolare, e di fosforo (P) i quantitativi di tossine prodotte sono inferiori. Tra i fattori che maggiormente influenzano la crescita e la tossicità di O. cf. ovata è emersa anche la competizione con altre microalghe (e.g. diatomee o dinoflagellate bentoniche), con effetti sulla crescita; sembrano inoltre essere esclusi possibili effetti allelopatici delle ovatossine verso le altre microalghe indagate. La comunità batterica associata ad O. cf. ovata in coltura sembra anch'essa interferire sulla produzione di tossine tramite re-mineralizzazione di sostanza organica, influenzando i quantitativi di N e P disponibile, e tramite interazioni di tipo mutualistico (Vanucci et al., 2012a). I risultati ottenuti evidenziano che da un lato lo stato fisiologico delle cellule è strettamente legato alle condizioni chimicofisiche e allo stato trofico del sistema e dall'altro questo si riflette sulla produzione e sul rilascio delle tossine. Cohu S., Mangialajo L., Thibaut T., Blanfuné A., Marro S., Lemée R., 2013. Harmful Algae 24, 32-44. Pezzolesi L., Guerrini F., Ciminiello P., Dell'Aversano C., Dello Iacovo E., Fattorusso E., Forino M., Tartaglione L., Pistocchi R., 2012. Water Research 46, 82-92. Pezzolesi L., Pistocchi R., Fratangeli F., Dell'Aversano C., Dello Iacovo E., Tartaglione L., 2014. Harmful Algae 36, 1-10. Pistocchi R., Pezzolesi L., Guerrini F., Vanucci S., Dell'Aversano C., Fattorusso E., 2011. Toxicon 57, 421-428. Rhodes L., 2011. Toxicon 57 (3), 400–407. Vanucci S., Guerrini F., Pezzolesi L., Dell'Aversano C., Ciminiello P., Pistocchi R., 2012a. Cryptogamie, Algologie 33, 105-112. Vanucci S., Pezzolesi L., Pistocchi R., Ciminiello P., Dell'Aversano C., Dello Iacovo E., Fattorusso E., Tartaglione L., Guerrini F., 2012b. Harmful Algae 15, 78